Project summary/Abstract: The family of Grb2-associated binding (Gab) scaffolding/adaptor proteins consist of a family of 3 highly conserved members (Gab1-3) that are expressed in a variety of host tissue cells including hematopoietic cells. Using a forward genetic approach in mice, we discovered that Gab3 is required for NK cell function. Gab proteins lack enzymatic activity and their function is defined by folded N-termini comprising a pleckstrin homology (PH) domain and C-terminal tails that harbor multiple phosphorylation sites for different tyrosine kinases. Following activation, Gab proteins are recruited to the plasma membrane through PH domain interaction with the membrane lipid phosphatidyl inositol-3,4,5-triphosphate (PIP3). Our compelling novel studies identify Gab3 as a critical mediator required for NK cell function. Using 2 independent Gab3-deficient mouse models, a functional hypomorph affecting PH domain function and a complete Gab3 KO model, we show that Gab3 is essential for recognition and elimination of ?missing-self? and tumor targets in vivo. At the cellular level, we found that IL-2/IL- 15-induced NK cell priming and expansion is impaired, and could be linked with a selective loss of IL-2/IL-15 MAPK but not STAT5 signaling. At the biochemical level, we identify the R27 residue (for which a human missense variant exists) as essential for optimal interaction with membrane lipid PIP2 and PIP3, suggesting that mechanistically a lack of recruitment of Gab3 downstream of IL-2 or IL-15 signaling may underlie the NK cell deficiency. The current application will test the hypothesis that Gab3 is a critical scaffolding protein required for expansion and function of NK cells downstream of IL-2/IL-15-signaling. Specifically, we posit that upon IL-2/IL- 15-induced activation of NK cells, Gab3 is recruited to the surface membrane, promoting a protein complex assembly that facilitates IL-2/IL-15 signaling in NK cells. The hypothesis will be tested in 2 specific aims. In the first aim, we will investigate the PIP binding specificity for human/mouse WT Gab3 and Gab3R27H or Gab3R27C missense mutants and define the scaffold protein network upon IL-2/IL-15 receptor activation in NK cells. The second aim will delineate the requirement for Gab3 in NK cell development, peripheral homeostasis and during infection. By revealing the specific role of Gab3 in NK cell function, our results will provide a new opportunity to selectively induce or repress NK cell function in settings of autoimmunity, anti-tumor immunity, transplantation, and other therapeutically important settings.